The evolution of the Aristolochia pallida complex (Aristolochiaceae) challenges traditional taxonomy and reflects large‐scale glacial refugia in the Mediterranean

Abstract The taxonomy of the Mediterranean Aristolochia pallida complex has been under debate since several decades with the following species currently recognized: A. pallida, A. lutea, A. nardiana, A. microstoma, A. merxmuelleri, A. croatica, and A. castellana. These taxa are distributed from Iberia to Turkey. To reconstruct phylogenetic and biogeographic patterns, we employed cpDNA sequence variation using both noncoding (intron and spacer) and protein‐coding regions (i.e., trnK intron, matK gene, and trnK‐psbA spacer). Our results show that the morphology‐based traditional taxonomy was not corroborated by our phylogenetic analyses. Aristolochia pallida, A. lutea, A. nardiana, and A. microstoma were not monophyletic. Instead, strong geographic signals were detected. Two major clades, one exclusively occurring in Greece and a second one of pan‐Mediterranean distribution, were found. Several subclades distributed in Greece, NW Turkey, Italy, as well as amphi‐Adriatic subclades, and a subgroup of southern France and Spain, were revealed. The distribution areas of these groups are in close vicinity to hypothesized glacial refugia areas in the Mediterranean. According to molecular clock analyses the diversification of this complex started around 3–3.3 my, before the onset of glaciation cycles, and the further evolution of and within major lineages falls into the Pleistocene. Based on these data, we conclude that the Aristolochia pallida alliance survived in different Mediterranean refugia rarely with low, but often with a high potential for range extension, and a high degree of morphological diversity.

Peninsulas, several glacial refugia have been identified for plants and animals (Feliner, 2014;Hewitt, 1999Hewitt, , 2011Médail & Diadema, 2009;Schmitt, 2007). The spatial and temporal dimensions of persisting series of Quaternary climate fluctuations show a high degree of complexity which may generate different biogeographic histories (Feliner, 2014;Gómez & Lunt, 2007). An example for a group with multiple refugial areas is the Euphorbia verrucosa L. alliance (Euphorbiaceae) which survived the ice ages in the Iberian, Apennine and Balkan Peninsulas (Cresti et al., 2019).
nardiana from A. merxmuelleri and A. pallida by sharing an elongated rootstock and A. microstoma by its unique perianth morphology.
Aristolochia pallida and A. merxmuelleri are characterized by a globose tuberous tuber.
First phylogenetic analyses by Wanke (2006) found A. pallida and A. lutea intermingled in one clade, as sister to A. merxmuelleri.
Accordingly, it was advised to sink A. lutea into A. pallida. The weakly supported sister clade to this group comprised A. microstoma and A. nardiana. In Wanke (2006) A. pallida and A. lutea, occurring from France to Turkey, were sampled with a very limited number of accessions. We here extent phylogenetic analyses with a comprehensive sampling of the A. pallida group covering its entire distribution range to address the following questions.
1. Are the recognized taxa in the A. pallida complex monophyletic? 2. Can lineages in the A. pallida group be interpreted as glacial relicts based on age estimations? 3. Do they correspond to geographic units on the regional scale and/ or in terms of hypothesized glacial refugia in the Mediterranean? 2 | MATERIAL S AND ME THODS

| Taxon sampling
Based on Wanke (2006) and Costa (2008), we included all currently accepted species of the A. pallida complex in our analysis (A. pallida, A. lutea, A. nardiana, A. microstoma, A. merxmuelleri, A. croatica, and A. castellana). In total, 87 accessions of the aforementioned taxa covering both the entire distribution range as well as the phenotypic diversity were included. The phenotypic diversity of our ingroup taxa is illustrated in Figure 1. The distribution of the A. pallida complex as well as the origin of the sampled accessions is shown in Appendix S1.
Additional 41 accessions of 33 Aristolochia taxa were selected to include the entire diversity of Aristolochia section Diplolobus (subsection Aristolochia and Podanthemum) as well as more distantly related outgroups from the remaining Aristolochia lineages (Wanke, 2006;Wanke et al., 2006Wanke et al., , 2007 in natural populations, respectively. DNA extraction was performed using a double-extraction approach with CTAB according to Borsch et al. (2003) or employing the extraction kit NucleoSpin Plant II (Macherey-Nagel, Düren, Germany) following the manufacturer's protocol. Several DNA markers were tested for the selected accessions. The amplification of nrITS using the standard primers ITS-A, ITS-B, ITS-C, and ITS-D (Blattner, 1999) was not successful.
PCR products were cleaned with the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany). If necessary, PCR products were purified using a 1.2% agarose extraction gel and the NucleoSpin Extract II-Kit (Macherey-Nagel). Both strands were obtained for each PCR product using the PCR primers and BigDye

| Divergence time estimation
To evaluate divergence times for the Aristolochia pallida complex, we used Bayesian inference (BI) implemented in BEAST v2.5 . As calibration we used fossils described from the late Miocene in Austria (Meller, 2014). These leaf impressions, described as Aristolochia austriaca Meller, are the most reliable paleontological records of the genus among the few Aristolochia fossils (Meller, 2014). They were found in the Hollabrunn-Mistelbach Formation which is dated to 11.0 or 11.1 Ma (Harzhauser et al., 2011;Roetzel et al., 1999). The author discussed similarities of these fossils and the extant A. rotunda and A. baetica. We followed this interpretation and used these dates in a first step dating analyses of  (Rambaut & Drummond, 2007). Due to low support values in several clades, we refrained from applying biogeographic ancestral area analyses.

| Haplotype network
The cpDNA dataset was analyzed through a statistical parsimony algorithm (Templeton et al., 1992), as implemented in TCS 1.21 (Clement et al., 2000), to infer genealogical relationships among haplotypes. The maximum number of differences resulting from single substitutions among haplotypes was calculated with 95% connection limit. Gaps were treated as missing data. The network was edited using tcsBU (Múrias dos Santos et al., 2016).

| Phylogenetic analyses and Haplotype network
Our study generated 99 new sequences of the trnK-matK-psbA region. The aligned sequence length was 4061 bp. The BI analyses showed rapidly converging chains and yielded trees with a mean log-  The ML and BI analyses resulted in two clades within the A.
pallida group (Figure 2). One is rather poorly supported (PP 0.79/ BS 78, but 0.99 PP in the Beast 2 analysis, Figure 6) and comprises

| Divergence time estimation
The

| Taxonomy
Our phylogenetic reconstruction of the A. pallida complex is based on the plastome trnk-matK-psbA region which has previously been suggested to provide good support and resolution on species level (Shaw et al., 2005). In our group, this marker shows low variation rates within a species complex but rather good resolution on species level in Aristolochia (Figure 2 Nardi, 1984). These morphotypes are dispersed throughout the phylogenetic tree and the haplotype network. The phylogenetic relationships argue for a high morphological variation within geographic subclades (Figure 4). The diverse flower morphology may be linked to the specialized pollination syndromes in this group (Rulik et al., 2008). Comparable results were found for the Turkish A. hirta group, indicating that morphological variation could have been conserved in refugial areas or evolved recently, also as result of hybridization (Mahfoud, 2010). Despite slight morphological differences, A. lutea, described in 1808, should be sunk into the earlier, in 1805 described Aristolochia pallida, confirming Wanke (2006). Because A. croatica shares the same haplotypes (group 9, Figure 3) as A. lutea and A. pallida the maintenance as its own species is not warranted.
Costa (2008)  from France. Therefore, its treatment as own species may be questioned.
Aristolochia merxmuelleri has been described on the basis of its morphological characters, color, and distribution (Mayer & Greuter, 1985;. It differs from other taxa of the A. pallida from the Balkan by the diminutive size, triangular to almost sagittate-reniform leaves, a pronounced hump on the back, the length of the flower peduncles, and an ovary which is longer than the petiole in fully developed plants. Indeed, the distinctiveness of A. merxmuelleri is supported also by our molecular data possessing own haplotypes and appearing a single clade (group 4) in the phylogenetic trees (Figures 2 and 3, Table 1). Previously, A. merxmuelleri was only known from serpentine areas in Kosovo.
Recent reports  indicate that A. merxmuelleri is present in northeast Albania. In its distribution range, A. merxmuelleri overlaps with other species of the A. pallida complex.
Therefore, a denser sampling in the future might breakup the monophylly of A. merxmuelleri as well.
Morphologically, the Greek A. microstoma can be clearly separated from all other species by unique fyke-shaped perianth with a very narrow entrance, flowers usually appear at ground level in the leaf-litter or between rocks (Rupp et al., 2021;Wanke, 2006). Our molecular data mainly confirm to treat A. microstoma as an evolutionary unit (group 2). Only one accession morphologically identified as A. nardiana (6), but occurring close to the area of distribution of A. microstoma, namely Euboea, is found inside the clade of A. microstoma (Nardi, 1991 Aristolochia nardiana has been delimited from A. pallida and A. lutea by its perianth shape and an elongated tuber (Nardi, 1989).
Although the aerial characteristics within A. nardiana seem to be stable, numerous A. lutea morphotypes from all over the distribution area of A. pallida s.l. show similar features. The tuber shape, long versus globose, as diagnostical character is relativized by the infraspecific variation in A. nardiana ranging from ellipsoid to slender shapes (Nardi, 1989). This shape may be linked to ecological parameters, for example, varying precipitation quantities in combination with different substrates. Similar observations have been found for A. rotunda s.l. where globose and elongate (subsp.
insularis) tubers occur throughout the southern distribution range (Nardi, 1991 to identify morphologically and would thus not help, for example, local floristic treatments and thus the general acceptance. We here refrain from formally drawing taxonomic conclusions given that until now only plastome-derived molecular markers were used and those results will need confirmation by nuclear-derived loci either substantiating our findings or providing an alternative evolutionary scenario.

| Biogeography
The evolutionary patterns of the A. pallida complex deviate from former taxonomic concepts (Figures 2-4). It opens the question whether the taxa may represent glacial relicts, an aspect that may have been disregarded when establishing morphology-based taxonomy. Based on our molecular clock analyses, the crown node of the A. pallida complex is dated to the Upper Pliocene, being about 3.0-3.3 Ma old, before the onset of glaciation cycles in the Pleistocene. The differentiation processes within the Greek and the pan-Mediterranean clades, however, fall into European glaciation times. Therefore, the temporal patterns support an interpretation that the evolutionary patterns were highly influenced by Pleistocene climate changes.
Our data contain a strong geographic signal (Figure 4), especially in longitudinal direction, which is a frequent pattern in Mediterranean taxa (Feliner, 2014). The Greek clade is exclusively distributed in Greece and haplotype networks including the sister group of the A. pallida complex identify this region as a potential source area for the entire group. Within the Greek clade (Figures 2 and 3, Table 1 within refugia" hypothesis by Gómez and Lunt (2007), originally developed for the Iberian peninsula. An East Mediterranean, for example, Anatolian, origin was also found for the stem node of Echium, Borago, and Anchusa s.l. clades of Boraginaceae by Mansion et al. (2009). Still, as Feliner (2014 pointed out, patterns of glacial refugia and range expansion in the Mediterranean are often very complex and different spatial and temporal levels need to be distinguished.
Using the present data, we restrict conclusions concerning the evolutionary history of the A. pallida group to larger geographical scales.
The pan-Mediterranean clade contains central and western Mediterranean and North Western Turkish groups. Within the A. pallida complex, A. pallida and A. lutea disintegrate into several supported geographic subclades. These subclades may likely represent regional radiations out of different glacial refugia in the Mediterranean. The Italian peninsula represented by A. lutea and A. pallida (group 8, Figure 3) occupies a central position in the haplotype network. This haplotype is closely related to several amphi-Adriatic groups mainly containing not only A. lutea but also A. pallida and A. croatica. Similar biogeographic patterns have been found for the amphi-Adriatic Campanula garganica Ten. clade (Campanulaceae; Park et al., 2006) and for Euphorbia myrsinites L. (Euphorbiaceae; Falch et al., 2019). Park et al. (2006) roughly estimated the origin of the disjunct Campanula lineage to the early to late Pleistocene.
This corresponds well to our molecular clock analyses dating these events also to the late Pleistocene. Northern Istria is well represented by A. lutea and A. croatica in our datasets. It is one of several hypothesized refugia areas for the Western Balkan (Médail & Diadema, 2009)   Aristolochia merxmuelleri (group 4, Figures 2 and 3) is clearly separated from other taxa, possibly since the early Pleistocene ( Figure 6). This may argue to interpret its limited present area of distribution in Kosovo and northeast Albania  as a long-term glacial refugium. A range extension of this stenoecious taxon may have been hampered by its specific ecological preference to a serpentine rocky substratum which is geographically restricted to few areas in the Western Balkans (Mayer & Greuter, 1985).
The westernmost distributed taxon is A. castellana with a narrow distribution in Central Spain (Costa, 2008). Costa (2008) proposed that A. castellana is a relict, paleoendemic taxon. This would imply that Iberia may have served as source area for a postglacial range extension in eastwards direction. In our analyses, this species has two exclusive haplotypes and one which is shared with southern French A. pallida (group 7, Figure 3). These haplotypes are closely related to Northern Italian A. pallida and A. lutea (group 8, Figure 3). Thus, our data reject the hypothesis that A. castellana is a paleoendemic relict. pallida group would require an even denser taxon sampling. Because these endemic rich areas are often found in Mediterranean mountains (Médail & Diadema, 2009;Noroozi et al., 2019) and members of the A. pallida complex are not restricted to high altitudes, survival during ice ages outside these classic areas, but in vicinity to them, seems possible. Hughes et al. (2006) (Feliner, 2014), including some subclades with limited dispersal capabilities at the regional scale and others with much wider range extension and some disjunctions where long distance dispersal may have been involved.

ACK N OWLED G M ENTS
We thank Anne-Kristin Schilling (

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.